Project Summary/Abstract The Hk1/Rrp1 Two component system (TCS) in Borrelia burgdorferi (Bb) is comprised of a membrane-bound hybrid sensory histidine kinase (Hk1) and a cytoplasmic response regulator with diguanylate cyclase activity (Rrp1). Bb lacking either Hk1 or Rrp1 are destroyed within ticks during the blood meal. In the current application, we will investigate how the sensory and effector components of the Hk1/Rrp1 TCS work together to promote survival of Bb in feeding ticks. In Aim One, we focus on the Hk1's three tandem periplasmic sensor SBP domains (D1-3). Using a site- directed mutagenesis approach, we will determine the contribution(s) of individual SBP domains to activation of Rrp1 in vitro and during the acquisition and transmission blood meals. To better understand how the Hk1 sensor domains function, we will build upon our success crystalizing D1 by solving the structures for D2, D3 and the full-length sensor. Results from structural analyses will help to refine the predicted ligand binding pockets for D2 and D3 and provide insight into how the three domains interface within the native HK. While there is consensus on the dramatic nature of the ?hk1/?rrp1 phenotype, we have only a limited understanding of how the output of this TCS, c-di-GMP, promotes survival of Bb in ticks. RNA-Seq and proteomic analyses of Rrp1- deficient organisms lead us to hypothesize that c-di-GMP signaling promotes metabolic and physiological adaptation to the blood meal as a means of evading killing by exogenous stressors generated during the feeding process. In our second Aim, we will test this hypothesis by manipulating in vitro conditions to more closely mirror those encountered by Bb in the fed midgut. In addition, we also will mutagenize select c-di-GMP-regulated cell envelope associated genes and phenotypically characterize the resulting mutants during acquisition and transmission blood meals. Our proposal will methodologically advance our understanding of how Bb perceives and responds to environmental signals encountered at the tick-mammal interface. Although beyond the scope of this proposal, our work has clear implications for the development of novel approaches to curtail Lyme disease, a rapidly expanding threat to public health.